PB2378: COMPREHENSIVE ANALYSIS OF P53-RESPONSIVE MIRNA’S GENES METHYLATION, TP53 GENE MUTATIONS AND POLYADENYLATION SIGNAL DESTRUCTION IN DIFFUSE LARGE B-CELL LYMPHOMA TUMOR TISSUE.
Authors: Elena Voropaeva, Tatyana Pospelova, Maria Churkina, Olga Berezina, Anna Gurazheva, Vladimir Maximov
Journal: HemaSphere (2023)

Topic: 20. Lymphoma Biology & Translational Research Background: Introduction of microRNA profiling revealed a decrease in the level of p53-responsive oncosuppressive microRNAs miR-34a, miR-34b, miR-34c, miR-129 and miR-203 in lymphoma tumor tissue in comparison with normal lymphoid tissue. It is known that mutations in the DNA-binding domain of the TP53 gene responsible for the decreased expression of these molecules not only due to the absence of transcription-dependent activation, but also due to slowing down the processing of microRNAs in the nucleus. Most microRNA genes are located in CpG-rich regions, however, data on the frequency of methylation of MIR-34A, MIR-34B/C, MIR-129-2 and MIR-203 genes in lymphomas are very scarce. Aims: The aim of this study was to conduct a comprehensive analysis of the methylation of the p53-responsive microRNA genes MIR-34A, MIR-34B/C, MIR-203 and MIR-129-2, mutations in the TP53 gene DNA-binding domain and impairment of the TP53 polyadenylation signal in diffuse large B-cell lymphoma (DLBCL). Methods: 136 DNA samples isolated from tumor tissue of patients with DLBCL and 11 DNA samples obtained from lymph nodes with reactive B-cell follicular hyperplasia were analyzed. The methylation status of MIR-203 and MIR-129-2 genes was determined by the method of methyl-specific PCR, MIR-34A and MIR-34B/C genes by the method of methyl–sensitive analysis of high-resolution melting curves. In tumor samples, destruction of the polyadenylation signal was performed by rs78378222 genotyping by polymerase chain reaction with restriction fragment length polymorphism, and the nucleotide sequence of the TP53 DNA-binding domain was determined by capillary direct sequencing by Sanger. A notion of the combined detection of the studied genes methylation was obtained using the OncoPrinter, the calculation of Log2 Odds Ratio and one-sided Fisher’s exact test (p-value) adjusted for multiple comparisons using the Benjamini-Hochberg procedure (q-value) was carried out. Results: The frequency of methylation of MIR-34A, MIR-34B/C, MIR-203 and MIR-129-2 and TP53 gene aberrations (mutations and destruction of the polyadenylation signal) was 23%, 55%, 66%, 65% and 21%, respectively. The detected methylation was tumor-specific. The absence of methylation of at least one of the studied microRNA genes occurred only in 15/136 (11.0%) cases of lymphoma, while in the vast majority of samples, methylation was combined. Thus, in 15/136 (11.1%) cases methylation of all 4, and in 60/136 (44.1%) - 3, and in 33/136 (24.3%) - 2 from the analyzed genes took place. At the same time it was shown that methylation of the analyzed p53-responsive microRNA genes and aberrations in the TP53 gene in the DLBCL tumor tissue were independent events (table). The analysis of MIR-34A, MIR-34В/С, MIR-203 and MIR-129-2 genes methylation and TP53 gene aberrations by the OncoPrinter - Gene methylation Gene aberrations p-value q-value Tendency MIR-34A TP53 0,297 0,424 Mutual exclusivity MIR-129-2 TP53 0,412 0,458 Mutual exclusivity MIR-34B/C TP53 0,264 0,424 Mutual exclusivity MIR-203 TP53 0,225 0,424 Mutual exclusivity Summary/Conclusion: Along with the TP53 gene aberrations tumor specific methylation may cause a decrease in the expression of miR-34a, miR-34b, miR-34c, miR-129 and miR-203 in DLBCL. It can be assumed that the combined methylation of the MIR-203, MIR-129-2 and MIR-34B/C genes, as well as the MIR-34B/C and MIR-34A pairs, potentially has a more pronounced pro-tumor effect due to the presence of common targets in the microRNAs encoded by them. Keywords: Gene mutation, Epigenetic, Diffuse large B cell lymphoma, TP53